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Immunology |
Department of Medical Physiology, Cardiovascular Research Institute, Texas A&M University System Health Science Center, College Station, Texas 77843-1114 [C-I. C., L. K.], and Department of Chemical Engineering, University of California, Los Angeles, California 90095-1592 [J. C. L.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In macrophages, L-arginine is metabolized by iNOS to form NO and L-citrulline. The antitumor activity of macrophages is found to be attributable to the production of NO3 (7) . In addition to the iNOS pathway, macrophages also express arginase which converts L-arginine into L-ornithine and urea (8, 9, 10, 11) . The resultant L-ornithine can be subsequently used by ODC to form polyamines (i.e., putrescine, spermidine, and spermine; 12 ), the essential nutrients required for the proliferation, differentiation, and neoplastic transformation of mammalian cells (13, 14, 15, 16) . Thus, it is likely that macrophages are capable of promoting tumor cell proliferation through the arginase pathway. Interestingly, in an animal tumor model, the increase in NOS activity was seen at the stage of tumor rejection, whereas the increase in arginase activity was observed during tumor growth (5) . These results suggest that L-arginine metabolism in macrophages at the tumor site, through the iNOS or arginase pathways, may have either detrimental or beneficial influence on the tumor growth, depending on which of the two pathways is prevailing. This contention is supported by the findings that the tumoricidal activity of macrophages was increased when transfected with the iNOS gene (7 , 17 , 18) . However, whether macrophage arginase can promote tumor proliferation and functionally counteract the NO-mediated cytotoxicity remains undetermined. In this regard, we investigated the role of macrophage arginase in tumor growth/cytotoxicity by overexpressing the arginase gene in macrophages. The effects of arginase overexpression on tumor cell proliferation and cytotoxicity were investigated in a coculture system.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Cultures.
The murine macrophage cell line J774A.1 and human breast adenocarcinoma
cell line ZR-75–1 were obtained from the American Type Culture
Collection (Rockville, MD). Cells were maintained in 60-mm culture
dishes with CDMEM supplemented with 1 mM sodium pyruvate, 2
mM L-glutamine, 100 units/ml penicillin, 100
µg/ml streptomycin, 0.25 µg/ml amphotericin B, 50 µg/ml
gentamicin, and 10% FBS (Summit Biotechnology, Fort Collins, CO) for
macrophages or 5% FBS for tumor cells. Cells were cultured at 37°C
under a humidified 10% CO2 atmosphere. Media
were changed daily, and cells were passaged after confluence by
trypsinization with DPBS containing 0.25% trypsin (Life Technologies,
Inc., Rockville, MD) and 0.02% EDTA. To study the interactions between
macrophages and tumor cells, a Falcon cell culture insert system
(Fisher Scientific Co., Pittsburgh, PA) was used. In this coculture
system, macrophages were grown in the cell culture inserts with a
membrane of 1.0-µm pore size, and the tumor cells were grown in the
bottom wells of the tissue culture plate.
Arginase Transfection.
Macrophages transfected with the rat liver arginase (arginase I) gene
were used to characterize the possible roles of macrophage arginase in
tumor growth. The arginase I cDNA (Ref. 19
; a gift from
Dr. Masataka Mori, Department of Molecular Genetics, Kumamoto School of
Medicine, Kumamoto, Japan) was cloned into the eukaryotic
expression plasmid pEGFP (Invitrogen Corp., Carlsbad, CA). All plasmid
DNA was prepared using a plasmid DNA preparation kit from Bio-Rad
Laboratories Inc. (Hercules, CA).
Macrophages were transfected using a DEAE-dextran method as described by Rupprecht and Coleman (20) with modifications. Before transfection, macrophages were plated at a density of 5 x 105 cells per insert and incubated overnight with CDMEM containing 10% FBS. Nonadherent cells were removed by gently washing the monolayer twice with DPBS, and the adherent cells were used for transfection. A DEAE-dextran stock solution was prepared in DPBS at a concentration of 20 mg/ml and filter sterilized. The transfection medium was then prepared by adding 1.5 ml of DMEM containing 80 µg/ml DEAE-dextran to 1.5 ml of DMEM containing 2 µg DNA of the expression plasmid (pEGFP) or the expression plasmid containing the arginase gene (pEGFP-Arg). The transfection medium was gently mixed at room temperature for 2 min and was added to the insert. After incubating the cells for 90 min at 37°C, the transfection medium was replaced with CDMEM containing 10% FBS. Arginase activity and its protein expression were then evaluated in nontransfected (control) and transfected (i.e., pEGFP and pEGFP-Arg) macrophages after a 24-, 36-, 48-, and 60-h incubation. In a separate set of experiments, cells were transfected with the ß-Gal gene to determine transfection efficiency using a ß-Gal staining kit (Invitrogen Corp.) following the manufacturer’s instructions.
Arginase Activity Assay and Immunoblotting of Arginase.
To prepare cell lysate for arginase activity assay and immunoblotting
analysis, cells were first rinsed with ice-cold DPBS twice after each
specified treatment and then scraped into 300 µl of lysis buffer
containing 50 mM Tris-HCl (pH 7.5), 0.1 mM
EDTA, 0.1 mM EGTA, 1 µg/ml leupeptin, 1 µg/ml
aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride.
Finally, cells were lysed by sonication at the frequency of 20 KHz
(Sonic & Materials, Inc., Danbury, Connecticut) for 30 s (10
s/cycle). Arginase activity in the cell lysates was measured as
described previously (21
, 22)
. In brief, cell lysate (50
µl) was added to 50 µl of Tris-HCl (50 mM; pH 7.5)
containing 10 mM MnCl2. Macrophage
arginase was then activated by heating this mixture at 55–60°C for
10 min. The hydrolysis reaction of L-arginine by arginase
was carried out by incubating the mixture containing activated arginase
with 50 µl of L-arginine (0.5 M; pH 9.7) at
37°C for 1 h and was stopped by adding 400 µl of the acid
solution mixture
(H2SO4:H3PO4:H2O = 1:3:7). For colorimetric determination of urea,
-isonitrosopropiophenone (25 µl, 9% in absolute ethanol) was then
added, and the mixture was heated at 100°C for 45 min. After placing
the samples in the dark for 10 min at room temperature, the urea
concentration was determined spectrophotometrically by the absorbance
at 550 nm measured with a microplate reader (Molecular Devices, Menlo
Park, CA). The amount of the urea produced was used as an index for
arginase activity.
Arginase protein was detected with the monoclonal antibody against human arginase I (Transduction Laboratories, Lexington, KY). The cross-reactivity of this antibody with rat arginase I has been demonstrated by the manufacturer. For immunoblotting of arginase I, cell lysates were prepared as described above, and equal amounts (25 µg) of proteins from each sample were subjected to a 10% SDS-PAGE. After electrophoresis, proteins were electrotransferred to nitrocellulose membranes (Bio-Rad Laboratories). The nitrocellulose membranes were incubated with the monoclonal primary antibody against arginase I and then with a horseradish peroxidase-conjugated antimouse IgG antibody. The proteins were visualized using the enhanced chemiluminescence detection kits (Amersham Pharmacia Biotech Inc., Piscataway, NJ).
Cell Proliferation Assay.
Tumor cells were seeded at a density of 1.2 x 105 per well of a 6-well culture plate and
incubated with CDMEM containing 0.5% FBS for 24 h. Inserts
containing nontransfected macrophages or macrophages at 24 h after
transfection (i.e., pEGFP or pEGFP-Arg) were then introduced
into the culture plate containing tumor cells. To carry out the
experiments under a more physiological level of
L-arginine, modified RPMI 1640 (Life
Technologies, Inc.) containing 100 µM
L-arginine, 4% FBS, 200
µM glutamine, 10 mM
HEPES, and the antibiotics as described above was used for the
coculture experiments. After a 36-h incubation, tumor cell
proliferation was determined using a cell proliferation assay kit
(CellTiter96 AQueous one solution; Promega Corp.,
Madison, WI). In some experiments, the transfected macrophages were
treated with L-norvaline (20
mM), a specific arginase inhibitor, at 6 h
after transfection to verify the effect of arginase. At 24 h after
transfection (i.e., 18-h treatment of
L-norvaline), the macrophages were used for
coculture experiments. L-Norvaline was not added
to the coculture medium to avoid any direct effects of
L-norvaline on tumor cell growth. The macrophage
arginase activity and tumor cell proliferation were determined after a
36-h incubation.
To study the direct effect of macrophage-derived polyamines on tumor cell growth, another series of experiments were performed using tumor cells pretreated with DFMO (1 mM; provided by Aventis Pharmaceuticals, formerly known as Hoechst Marion Roussel, Inc., Bridgewater, NJ), a suicide inhibitor of ODC, for 24 h to arrest polyamine synthesis. The contents of L-arginine, L-ornithine, and polyamines (e.g., putrescine, spermine, and spermidine) in macrophages, tumor cells, and the culture media before and after coculture experiments were then measured with high-performance liquid chromatography.
Polyamines, L-Ornithine, and L-Arginine
Measurement.
The cell culture media were collected (1 ml) and the macrophages and
tumor cells were harvested by scraping into 300 µl of DPBS. The cells
were lysed with sonication at the frequency of 20 KHz for 30 s (10
s/cycle). Both the cell lysates and coculture media samples were
centrifuged at 12,000 x g for 5 min to
sediment cell debris and proteins, and the supernatant was passed
through the ultra-free filtration units with 5,000 molecular
weight cutoff (Millipore Corp., Bedford, MA). The polyamines and amino
acids were separated by a C-18 reverse phase-packed column (model
Hypersil AA-ODF; Hewlett Packard) and detected with an UV detector
after precolumn derivitization of the hydrolyzed primary amino acids or
polyamines with o-phthalaldehyde (OPA). The concentrations
of polyamines, L-arginine, and
L-ornithine in these supernatants were then
quantified as their OPA derivatives by the AminoQuant System (Hewlett
Packard, model HP 1090 L).
Cytotoxicity Assay.
At 24 h after transfection, macrophages were treated with LPS (1
µg/ml) for 8 h to induce their cytotoxic activity. The activated
macrophages were then cocultured with tumor cells in the RPMI medium
containing 50 µM L-arginine. The activity of
LDH released by the killed or dying tumor cells in the coculture medium
was measured with a cytotoxicity assay kit (CytoTox96, Promega Corp.)
and was used as an indicator for tumor cytotoxicity. The percentage
cytotoxicity was obtained using the following formula provided by
Promega Corp.
The level of NO in the coculture medium was determined using a chemiluminescence assay as described in our previous study (21) . In some experiments, a NOS inhibitor, L-NMMA (10 µM), was added together with LPS to confirm the specific effects of NO. In another series of studies, macrophages were treated with L-norvaline (20 mM) at 14 h after the transfection (i.e., for an 18-h incubation before coculture) to examine the role of arginase in counteracting NO-mediated cytotoxicity.
Protein Determination.
Proteins from each plate of cells were determined by bicinchoninic acid
protein assay (Pierce Chemical Co., Rockford, IL). Results from all of
the experiments are expressed on the basis of cell proteins.
Statistical Analysis.
Results are given as mean ± SE for at least three
independent experiments performed in triplicate. The statistical
significance of differences between groups was analyzed by one-way
ANOVA followed by Fisher’s protected least-significant differences
test using StatView 4 (Abacus Concepts, Berkeley, CA). A
P < 0.05 was considered to be statistically
significant.
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RESULTS |
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shows that nontransfected and pEGFP-transfected macrophages
had low levels of arginase activity (i.e., urea production)
that did not change with time. On the other hand, macrophages
transfected with pEGPF-Arg exhibited a time-dependent increase in
arginase activity. The increase was observed at 24 h after
transfection and reached the maximum (
5-fold increase) at 36 h
after transfection. Arginase activity remained elevated for up to
48 h and then decreased at 60 h after transfection (Fig. 1A)
. Arginase protein was below the detectable range in both
control and pEGFP-transfected macrophages, but its expression was
significantly increased after transfection with pEGFP-Arg. The
expression of arginase at 36 h after transfection of pEGFP-Arg is
shown in Fig. 1B
. A transfection efficiency assay carried
out at 36 h after transfection indicated that about 14 ± 2% of cells were transfected. Because the arginase activity
started to increase at 24 h after transfection, macrophages at
this stage were used in the following coculture studies.
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. Macrophages transfected with pEGFP did not increase arginase activity
and had no effect on tumor cell proliferation. On the other hand,
transfection of pEGFP-Arg significantly increased arginase activity and
enhanced proliferation of the tumor cells by 42% (Fig. 2A)
.
Pretreating the pEGFP-Arg-transfected macrophages with an arginase
inhibitor L-norvaline significantly inhibited
arginase activity and concomitantly reduced the growth of cocultured
tumor cells (Fig. 2A)
. These results indicate that
overexpression of macrophage arginase enhances tumor cell
proliferation. However, the observed overall tumor cell growth in this
set of studies resulted from the contribution of both exogenously
(i.e., from macrophages)- and endogenously (i.e.,
from tumor cells per se)-synthesized polyamines. To exclude
the contribution of endogenously synthesized polyamines, tumor cells
were pretreated with DFMO, and the effect of macrophage arginase on
tumor cell growth was reexamined. As shown in Fig. 2A
,
overexpression of arginase in macrophages enhanced the growth of
DFMO-treated tumor cells by 82%. L-Norvaline
inhibited arginase activity in pEGFP-Arg-transfected macrophages (Fig. 2B)
and also reduced their ability to promote tumor cell
growth.
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shows that L-arginine concentration
in the medium was significantly reduced from 100 nmol/ml (Fig. 3A
, Medium only) to 30 nmol/ml as tumor cells
were cocultured with either nontransfected (Fig. 3A
,
Control) or pEGFP-transfected (Fig. 3A
,
pEGFP) macrophages. In the presence of pEGFP-Arg-transfected
macrophages, L-arginine concentration in the
medium was further reduced to 18 nmol/ml (Fig. 3A)
. This
reduction was prevented by treating the pEGFP-Arg-transfected
macrophages with L-norvaline. The intracellular
level of L-arginine was not altered either by
arginase transfection or by L-norvaline (Fig. 3B)
.
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. Intracellular L-ornithine was also
significantly increased in the macrophages transfected with the
arginase gene. Treating the transfected macrophages with
L-norvaline not only reduced the
L-ornithine in the media but also reduced the
intracellular levels of L-ornithine in
macrophages (Fig. 4B)
. The L-ornithine
content in the tumor cells was consistently lower than that in the
macrophages, but its level was significantly increased with the
presence of the pEGFP-Arg-transfected macrophages (Fig. 4B)
.
This increase was prevented by treating the macrophages with
L-norvaline (Fig. 4B)
.
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. Similarly, the intracellular putrescine contents were
significantly increased in the arginase-transfected macrophages and the
corresponding cocultured tumor cells (Fig. 5B)
. Again,
pretreating the macrophages with L-norvaline
abolished the effect of arginase overexpression (Fig. 5)
. On the other
hand, spermine and spermidine were not detectable in either cell type
regardless of treatments.
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. Inhibition of NO synthase (by
L-NMMA) also had no effect on arginase activity.
In terms of NO production, the nonactivated macrophages
(i.e., without LPS treatment) released only a negligible
amount of NO (i.e., nitrite measurement) in the cocultured
medium (Fig. 6B)
. Under these conditions, the macrophages
exerted about 8% cytotoxicity toward tumor cells. LPS elicited a high
amount of NO production in the vector-transfected cells and produced a
3-fold increase in tumor cytotoxicity. The increased NO production and
tumor cytotoxicity were attenuated by L-NMMA.
Transfection of pEGFP-Arg into macrophages also attenuated the
LPS-induced NO production as well as the tumor cytotoxicity, whereas
L-norvaline partially reversed these inhibitory
effects (Fig. 6B)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, the correlation between tumor cell proliferation and macrophage arginase activity implicates the beneficial role of macrophage arginase in tumor growth, presumably through the production of L-ornithine and, thus, polyamines. Because arginase uses L-arginine as the substrate for L-ornithine synthesis, it is expected that the consumption of L-arginine would be increased in the presence of macrophages overexpressing arginase. Indeed, a significant decrease in the medium L-arginine in the coculture system was found, which indicated a high consumption rate of L-arginine by cultured cells, and this reduction in extracellular L-arginine was more pronounced in the presence of pEGFP-Arg-transfected macrophages. Treating the pEGFP-Arg-transfected macrophages with the arginase inhibitor L-norvaline prevented the reduction in L-arginine content in the medium, suggesting a direct result of arginase overexpression in this event. However, the levels of intracellular L-arginine remained unchanged in both macrophages and tumor cells regardless of different interventions, which implied that these cells mainly use exogenous L-arginine to maintain the homeostasis of an intracellular pool. This finding agrees with a previous study by Hrabák et al. showing that macrophage arginase mainly uses extracellular L-arginine, although the level of L-arginine within these cells was not reported (23) .
In contrast to L-arginine, the L-ornithine concentration in the coculture medium was elevated, particularly in the presence of macrophages transfected with the arginase gene. In addition, the L-ornithine level in both macrophages and tumor cells was also increased as a result of arginase overexpression in macrophages. Because the increase in L-ornithine level was abolished by pretreating macrophages with L-norvaline and the arginase activity in tumor cells was not altered during the coculture experiments (data not shown), we conclude that the increased L-ornithine in tumor cells is derived from the macrophage arginase. Interestingly, the L-ornithine level in macrophages was found to be consistently higher than that in the tumor cells, which may be attributable to the 3- to 4-fold higher arginase activity generally found in control macrophages than in tumor cells (data not shown). The low arginase activity and low L-ornithine level in tumor cells may also imply that they are heavily dependent on the exogenous supply of L-ornithine. As mentioned earlier, L-ornithine is converted to putrescine by a reaction catalyzed via ODC, thus the increased L-ornithine may contribute to the tumor cell growth through polyamine formation.
It should be noted that in the present study, the tumor cells were pretreated with DFMO, a suicide inhibitor for ODC that prevents the conversion of L-ornithine to putrescine. Therefore, these tumor cells were incapable of using L-ornithine for polyamine synthesis. This contention was supported by our study that showed DFMO completely blocked ODC activity in tumor cells as indicated by the depletion of endogenous polyamines. In this regard, it is believed that the observed increased tumor growth in the present study is a result of the increased availability of exogenous polyamines provided by the macrophages overexpressing arginase. Although three main polyamines, i.e., putrescine, spermidine, and spermine are found in mammalian cells, only putrescine was detected in macrophages in our study. This result is consistent with the findings of Boutard et al. that macrophages produced mainly putrescine (6) . The putrescine content in the coculture medium and in the macrophages as well as the tumor cells was significantly increased when the pEGFP-Arg-transfected macrophages were present. Interestingly, the amount of putrescine in the tumor cells was generally higher than that in macrophages, which suggests that these tumor cells are capable of taking up putrescine from the extracellular source (16 , 24) . The ability of tumor cells to use exogenous polyamines may also explain why ODC inhibitor DFMO can potently deplete polyamines and thus inhibit cells growth in most cell types in a mono-cell type culture system, but was ineffective in animal models (25 , 26) or in a coculture system as shown in the present study. Although we did not measure the transport of putrescine in tumor cells, our results indicate that arginase overexpression leads to a higher production of putrescine in macrophages. It appears that the putrescine released into the coculture medium is subsequently taken up by the tumor cells for their growth because blockade of arginase activity in macrophages prevented the increase of putrescine in both the coculture medium and tumor cells and consequently attenuated tumor cell proliferation.
In addition to arginase, the other main
L-arginine-consuming enzyme iNOS is expressed in activated
macrophages, and the generated NO is known to mediate the cytotoxic
effects of macrophages (7
, 27, 28, 29)
. Our results show that
a high amount of NO production in macrophages, after LPS-activation,
was accompanied with high tumor cytotoxicity. Interestingly, the
nonactivated macrophages also exhibited some degrees of tumor
cytotoxicity (8%). This basal cytotoxic effect is independent of NO
because the release of NO from resting macrophages is negligible, and
this basal cytotoxicity is insensitive to a NOS inhibitor
L-NMMA (data not shown). It is likely that the release of
reactive oxygen species and proteases by macrophages (2
, 3)
in response to "irritating" products released from tumor
cells (1)
accounts for this basal cytotoxicity.
Nevertheless, the increased tumoricidal effect of LPS-activated
macrophages was abolished by L-NMMA, indicating the
important role of NO in tumoricidal process. Overexpression of arginase
in macrophages resulted in a reduction of NO production and also
attenuated the tumor cytotoxicity. These effects were reversed by the
arginase inhibitor L-norvaline, which suggests the direct
role of arginase in regulating NO production and thus the tumoricidal
activity of activated macrophages. The down-regulation of NO production
by arginase might be a result of substrate competition,
e.g., reduction of L-arginine
availability to iNOS by the up-regulated arginase (21)
. We
have previously shown that the effect of arginase on NO production is
more pronounced when the extracellular supply of
L-arginine is limited (21)
.
Therefore, in the present study, the reduced
L-arginine content in the medium, caused by
arginase overexpression, might further enhance the counteraction of
arginase on the tumoricidal function of macrophages. Although a
decreased extracellular level of L-arginine was
commonly observed in wounds and during septic shock
(30, 31, 32)
, the L-arginine level at
the sites of tumors has not yet been determined. It is expected that
the change in L-arginine availability will
significantly influence the biological function of macrophages.
Together, our results suggest that the tumoricidal and growth-promoting activities of macrophages are regulated by the L-arginine metabolism through the iNOS and arginase pathways. Because both arginase and iNOS pathways have been shown to be up-regulated under certain pathophysiological conditions (5 , 6 , 33, 34, 35) , macrophages can therefore have very different functions in tumor growth, depending on which of the two pathways is prevailing. However, it is not yet known what factor, or combination of factors, is responsible for the regulation of iNOS or arginase activities in macrophages at tumor sites. Nevertheless, a recent study has shown that transforming growth factor–ß can attenuate the macrophage-mediated tumor cytolysis by down-regulating iNOS activity, although enhancing arginase activity in macrophages (6) . In addition, several cytokines such as IL-4, IL-10 (33 , 35) , and IL-13 (36) have been reported to induce arginase activation in macrophages. However, the involvement of these cytokines in the regulation of tumor cytotoxicity/growth remains to be elucidated. Furthermore, it has been shown that arginase activity can be inhibited by NG-hydroxy-L-arginine, an intermediate of the NOS pathway (37) . The cross-regulation between the NOS and arginase pathways further amplifies the complexity of the regulatory mechanism of L-arginine metabolism toward NO and polyamines. Understanding the regulation of iNOS and arginase activation and their reciprocal interaction may provide novel information for controlling tumor growth.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Heart, Lung, and Blood
Institute Grants KO2-HL-03693 (Research Career Award) and HL-55524 (to
L. K.). 
2 To whom requests for reprints should be
addressed, at Department of Medical Physiology, Cardiovascular Research
Institute, Texas A&M University System Health Science Center, College
Station, TX 77843-1114. Fax: (979) 847-8635; E-mail: lkuo{at}tamu.edu
3 The abbreviations used are: NO, nitric oxide;
iNOS; inducible NO synthase; ODC, ornithine decarboxylase; DFMO,
-difluoromethylornithine; LPS, lipopolysaccharide; L-NMMA,
NG-monomethyl-L-arginine; IL, interleukin;
CDMEM, complete DMEM; FBS, fetal bovine serum; DPBS, Dulbecco’s PBS;
LDH, lactate dehydrogenase.
Received 6/12/00. Accepted 11/27/00.
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S. El-Gayar, H. Thuring-Nahler, J. Pfeilschifter, M. Rollinghoff, and C. Bogdan Translational Control of Inducible Nitric Oxide Synthase by IL-13 and Arginine Availability in Inflammatory Macrophages J. Immunol., November 1, 2003; 171(9): 4561 - 4568. [Abstract] [Full Text] [PDF]
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P. C. Rodriguez, A. H. Zea, J. DeSalvo, K. S. Culotta, J. Zabaleta, D. G. Quiceno, J. B. Ochoa, and A. C. Ochoa L-Arginine Consumption by Macrophages Modulates the Expression of CD3{zeta} Chain in T Lymphocytes J. Immunol., August 1, 2003; 171(3): 1232 - 1239. [Abstract] [Full Text] [PDF]
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Y. Liu, J. A. Van Ginderachter, L. Brys, P. De Baetselier, G. Raes, and A. B. Geldhof Nitric Oxide-Independent CTL Suppression during Tumor Progression: Association with Arginase-Producing (M2) Myeloid Cells J. Immunol., May 15, 2003; 170(10): 5064 - 5074. [Abstract] [Full Text] [PDF]
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V. Bronte, P. Serafini, C. De Santo, I. Marigo, V. Tosello, A. Mazzoni, D. M. Segal, C. Staib, M. Lowel, G. Sutter, et al. IL-4-Induced Arginase 1 Suppresses Alloreactive T Cells in Tumor-Bearing Mice J. Immunol., January 1, 2003; 170(1): 270 - 278. [Abstract] [Full Text] [PDF]
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P. C. Rodriguez, A. H. Zea, K. S. Culotta, J. Zabaleta, J. B. Ochoa, and A. C. Ochoa Regulation of T Cell Receptor CD3zeta Chain Expression by L-Arginine J. Biol. Chem., June 7, 2002; 277(24): 21123 - 21129. [Abstract] [Full Text] [PDF]
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G. Raes, P. De Baetselier, W. Noel, A. Beschin, F. Brombacher, and G. Hassanzadeh Gh. Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated macrophages J. Leukoc. Biol., April 1, 2002; 71(4): 597 - 602. [Abstract] [Full Text] [PDF]
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A. P. Gobert, D. J. McGee, M. Akhtar, G. L. Mendz, J. C. Newton, Y. Cheng, H. L. T. Mobley, and K. T. Wilson Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: A strategy for bacterial survival PNAS, November 20, 2001; 98(24): 13844 - 13849. [Abstract] [Full Text] [PDF]
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, significantly
different from pEGFP-Arg at 24 h (P < 0.05). B, Western blot analysis of arginase
expression at 36 h after transfection. Arginase protein was not
detectable in nontransfected (control) and
pEGFP-transfected (pEGFP) cells, but was detected in
macrophages transfected with pEGFP-Arg. Protein from rat liver cells
was used as a positive control and each lane was loaded with an equal
amount (25 µg) of protein.
, non-DFMO-treated; 
, DFMO-treated) at the end of
the coculture. B, arginase activity in macrophages
cocultured with non-DFMO-treated tumor cells (
